In eukaryotes, protein synthesis for nearly all proteins begins in the cytoplasm via mega protein complexes called ribosomes. Various proteins complete their synthesis and folding in the cytoplasm and remain there where they function. However, many others are exported out of the cytoplasm and into the endoplasmic reticulum (ER) where they acquire the needed post-translational modifications (e.g., disulfide bonds, glycosylation, etc.) to attain their proper protein structure and biological activity prior to export to their intended cellular locations (e.g., Golgi, peroxisome and lysosomal proteins) or to the cell surface (e.g., receptors, ion channels, etc.) or secreted out of cells (e.g., antibodies, clotting factors, hormones, etc.). Proteins destined for export out of the cytoplasm are distinguished from cytoplasmic proteins by a specialized protein element at the amino (N-) terminus called the signal sequence.
Signal sequences (also called signal peptides) have no consensus amino acid sequence or length but typically comprise the initial 15-40 residues at the N-terminus with 7-20 contiguous hydrophobic amino acid residues which form an α-helical secondary structure that is often flanked by charged residues. Signal sequences are identified in the cytoplasm by a specialized multi-subunit protein:RNA complex called the signal recognition particle (SRP) which directs these nascent proteins to specialized pores within the ER membrane called translocons where these proteins are transported across the ER membrane into the ER lumen—a process known as protein translocation.
Protein translocation occurs concurrently during protein synthesis (i.e., co-translationally) in mammals while in other eukaryotes (e.g., yeast), this process can be either co- or post-translational. Signal sequence-mediated protein translocation is also utilized in bacteria for directing proteins out of the cytoplasm and into the periplasm. In mammals, signal sequences are identified by SRP as they emerge from ribosomes which temporarily pauses protein translation to allow the targeting of the entire SRP-nascent protein-ribosome complex to translocons via the associated SRP receptor. Protein synthesis is resumed after SRP is released and the ribosome-nascent protein complex is properly docked at the translocon.
Most enzyme and other protein therapeutics are produced by recombinant technology that is designed to secrete these recombinant proteins out of cells and into cell culture to simplify downstream purification. These recombinant enzymes and other proteins therefore must utilize signal sequences and this same cellular pathway for secretion. High-level production of these proteins therefore requires signal sequences that can mediate efficient ER targeting and protein translocation across the ER membrane. However, signal sequences are not equivalent for facilitating ER targeting and translocation. The identification of signal sequences by SRP is believed to occur rapidly and efficiently, but the subsequent ER targeting and translocation steps are highly disparate among proteins. Because signal sequences are recognized twice, first by SRP for targeting the nascent protein-ribosome complex to ER and subsequently by translocon proteins (i.e., Sec61 proteins) and other translocon-associated ER proteins to initiate translocation, both are potential sites for regulation. This latter step has been shown to be much more stringent and less efficient and thus, is a major bottleneck in this process. Surprisingly, most signal sequences are intrinsically inefficient for facilitating protein translocation. Consequently, many ER-targeted nascent protein-ribosome complexes dissociate from the ER membrane and protein synthesis is aborted, thereby reducing their protein expression and secretion.